Tenderization of meat with proteolytic enzymes

ABSTRACT

Sulfhydryl proteases such as papain, bromelain or ficin are treated with certain disulfide inactivators to reversibly inacitivate the active enzyme fraction while preserving essentially 100% of the total enzymatic activity. The inactivated enzyme is then injected ante-mortem into an animal&#39;&#39;s vascular system wherein the enzyme is reactivated at a controlled rate by naturally occurring reducing agents present in the animal&#39;&#39;s blood.

United States Patent 1191 1111 3,818,106 Kang et al. June 18, 1974 [5 TENDERIZATION 0F MEAT WITH 3,539,451 11/1970 Heinicke 195/68' PROTEOLYTIC ENZYMES 3,709,790 1/1973 Beuk et a1 99/107 X Inventors: ChungheeKim Kang, Hinsdale;

William D. Warner, Elmhurst; Eldon E. Rice, Palos Park, all of I11.

Assignee: Swift & Company, Chicago, Ill.

Filed: Oct. 2, 1972 Appl. No.: 294,205

U.S. Cl. 426/2 Int. Cl. A22c 18/00 Field of Search 99/107; 195/68; 426/2 References Cited UNITED STATES PATENTS v 9/1962 Hogan 99/l07 Primary Examiner-Hyman Lord Attorney, Agent, or Firm-Edward T. McCabe 5 7] ABSTRACT Sulthydryl proteasessuch as papain, bromelain or ficin are treated with certain disulfide inactivators to reversibly inacitivate the active enzyme fraction while preserving essentially 100% of the total enzymatic ac- 20 Claims, No Drawings TENDERIZATION OF MEAT WITH PROTEOLYTIC ENZYMES The present invention relates to the preparation of proteolytic enzyme formulations for tenderization of meat by ante-mortem injection. More specifically, the invention pertains to a process for preparing proteolytic enzyme formulations containing large amounts of reversibly inactivated enzyme molecules which are stable during storage, are slowly reactivated in the animal, and which produce minimal adverse animal reaction.

The technique of ante-mortem enzyme injection as a means of meat tenderization is disclosed and claimed in US. Pat. No. 2,903,362, issued Sept. 8, 1958. Briefly, this technique involves the injection of a solution of a proteolytic enzyme into the vascular system of a living animal, then holding the animal for atime sufficient to obtain unifonn distribution of the enzyme throughout the animals system, and thereafter slaughtering the animal. Meat cuts derived from animals injected in this manner are very tender and exhibit good textural properties. While this technique is of substantial value in obtaining meat which, when cooked, is uniformly tender, there are some undesirable animal side reactions which occur if commercially prepared enzyme formulations are employed. Specifically, injection of the enzyme into a live animal often causes internal hermorrhaging and edema of theinteral organs. As a result, the animal carcass would be condemned.

It has generally been thought that the adverse physiological reactions in live animals were primarily due to the presence of certain undesirable components (impurities) present in the commercially available enzyme. Much work has been done to remove or render harmless these undesirable components from commercial enzyme preparations. For example, US. Pat. No. 3,052,551 describes several methods for the purification and/or stabilization of enzyme solutions.

Unfortunately, these purification and/or stabilization procedures result in the irreversible inactivation of a substantial portion of the active enzyme. Since only active enzymes can tenderize meat, the irreversible inactivation of a substantial portion of the active enzyme results in an inefficient product. Work was therefore conducted on methods of reversibly inactivating the proteolytic enzymes without reducing total enzymatic activity. For example, US. Pat. No. 3,446,706 describes a method for preparing improved enzyme solutions comprising adjusting the pH of a solution to greater than 7.5, reversibly inactivating the solution by a hydrogen peroxide-catalase treatment, and then holding the solution at a pH of above 7.5 for from 2-24 hours. Although this technique produces a proteolytic enzyme formulation having improved activity, treatment of the enzyme with hydrogen peroxide still causes irreversible inactivation of a portion of the originally active enzymes.

R. N. Heinicke, in U.S. Pat. No. 3,539,451, described a method for reversibly inactivating the enzyme fraction of fresh papaya or fig latex, comprising treating the fresh latex with sodium tetrathionate at a pH in the range of 6.5-8.0. The reversible inactivation with tetrathionate is said to result in an activatable enzyme in high yield. Heinicke indicates in his patent, and particularly in his parent application, Ser. No. 693,612, filed Dec. 26, 1967 (now abandoned), that tetrathionate is the only disulfide reagent which will reversibly inactivate proteolytic enzymes without reducing total enzymatic activity, and which will result in an enzyme formulation that does not cause adverse physiological reactions when injected into live animals.

It is therefore an object of the present invention to provide additional reagents for the reversible inactivation of proteolytic enzymes for ante-mortem injection of animals.

It is another object of the present invention to provide an improved method of preparing reversibly inactivated enzyme formulations for ante-mortem injection of animals.

It is a further object of the present invention to provide an improved method for preparing reversibly inactivated enzyme solutions which are characterized by the retention of essentially of their enzymatic activity.

It is also an object of the present invention to provide an improved method of preparing reversibly inactivated proteolytic enzyme formulations which will not cause adverse physiological reactions upon antemortem injection into animals.

Additional objects of the present invention, if not specifically set forth herein, will be readily apparent to those skilled in the art from a reading of the following I detailed description of the invention.

We have discovered that, contrary to the work of Heinicke, there are several disulfide reagents,,other than sodium tetrathionate, which can be reacted with I ciently slow reactivation rates such that adverse physiological reactions in the injected animals is avoided.

Generally, the method of this invention comprises treating a sulfhydryl protease, held at a low temperature and at a pH of 6-12, with one or more of certain disulfide reagents that reversibly inactivate the active enzyme fraction. The reversibly inactivated enzyme may then be injected ante-mortem into animal stock. The invention is directed to the sulfhydryl proteases, i.e. proteases requiring a free sulfhydryl group for its catalytic activity, and is particularly directed to the plant sulfhydryl proteases papain, bromelain and ficin. Commercial enzyme powders of these plant derived enzymes contain a mixture of sulfliydryl proteases. For example, commercial papain contains pure papain enzyme, along with two or more chymopapain enzymes. In addition to the plant sulfhydryl proteases, this invention also contemplates the use of microbial sulfhydryl proteases such as streptococcal proteinase and clostripain, as well as animal sulfhydryl proteases such as cathepsin C. Commercial food grade enzyme powders contain mixtures of active" enzyme molecules, reversibly inactive enzyme molecules, and irreversibly inactive enzyme molecules. The ratios of these three types of molecules vary from one batch of enzyme to another.

Active enzyme molecules produce the meat tenderization through proteolysis, but also may cause severe physiological reactions when injected into live animals. For example, intravascular injection of live animals with crude enzyme solutions of either papain, bromelain, ficin, or combinations thereof, containing a substantial portion of active enzyme molecules, produce such symptoms as labored breathing, nasal congestion, depression, frothing at the mouth, and in severe cases the animal will become cyanotic and die. The autopsy findings of such reactors usually include hemorrhaging in the kidneys, heart, liver, intestines, gall bladder, and larynx. As a result of these symptoms, the reactors are condemned by governmental inspectors. Fully active enzymes may cause very rapid expiration of the animal.

lrreversibly inactive enzyme molecules are those which, due to oxidation, hydrolysis, etc., of the active enzyme molecule, have permanently lost their activity, at least under the processing conditions of antemortem enzyme solutions, and therefore produce no tenderization effect or animal reaction.

Reversibly inactive enzyme molecules appear to produce no tenderization or adverse animal reactions upon intravascular injection. However, these enzymes may be reactivated within the animals vascular system, thereby producing subsequent tenderization of the meat. Therefore, it is most desirable in methods of ante-mortem meat tenderization by intravascular injection to inject an enzyme solution containing as high a concentration of reversibly inactive enzyme molecules as is possible. That is to say, the problem of eliminating animal reaction is to reversibly inactivate the active enzyme molecules in such a manner that the enzyme will be reversibly inactivated at the time of injection, and will not be reactivated between injection and slaughter, or at least will be reactivated at a slow rate that can be tolerated by the animal.

An accepted method for developing a quantitative determination as to the enzyme activity of active molecules andas to the total enzyme activity, which is made up of both the active enzyme molecules and the reversibly inactive enzyme molecules, is set forth in a patent toMcAnally et al., U.S. Pat. No. 3,446,626. Essentially, the amount of active enzyme molecules in a given solution is determined by employing a milk clot assay of the available enzyme activity. The units for measuring active enzyme molecules are generally called non-reduced milk clot units (N-RMCU), and are sometimes referred to as the available milk clot activity (AMCU). Where a measure of the reversibly inactive enzyme molecules is desired, a second portion of the enzyme solution is treated with an activator or reducing agent such as cysteine, cyanide, bisulfite or the like, whereby all the reversibly inactivated enzyme is restored to the active state. The milk clot assay using added cysteine or other activators measures both the active and the reversibly inactive enzyme molecules. This measurement is referred to as either Reduced Milk Clot Units (RMCU) or Total Milk Clot Activity Units (TMCU). Any reduction in this value during processing indicates a loss in the overall enzyme activity of the solution being assayed. However, reduction in the N-RMCU without a corresponding reduction in the RMCU value, indicates the amount of active enzyme molecules which have been converted to reversibly inactivated molecules.

As has been previously mentioned, it was formerly believed by those skilled in the art that adverse animal reactions upon injection of proteolytic enzymes was due in part to impurities caused by enzyme autolysis and to the presence of impurities in the enzyme formulation, and specifically non-enzyme proteins and low molecular weight compounds. We have discovered however that with proteolytic enzymes, the animal reaction is apparently caused solely by the active enzyme per se. That is, when the enzyme is purified and then activated, it causes severe reaction when injected into animals. Even though most of the non-enzyme protein and low molecular weight compounds have been removed, the purified active enzyme causes as severe a reaction as an equivalent amount of crude enzyme. Thus no amount of purification of an active enzyme will eliminate animal reaction. In view of these discoveries, it became apparent that to obtain truly efficient enzyme formulations for ante-mortem injection, an inactivating reagent must meet all of the following requirements: l it must reversibly inactivate proteolytic enzyme, such that the enzyme will be inactive at the time of ante-mortem injection; (2) it must provide a modified enzyme that will reactivate in situ at such a rate that active enzyme will not be released between injection and slaughter so as to cause adverse physiological reactions, but active enzymes for tenderization will be released between slaughter and consumption of the meat; (3) it must not be toxic to humans at the levels that it will be present in the tenderized meat.

We have discovered that several low molecular weight, symmetrical disulfide compounds meet the above three criteria, and therefore have utility in the ante'mortem injection of animals with proteolytic enzymes. ln addition to sodium tetrathionate, suitable disulfide compounds in this class include 2-hydroxyethyl disulfide, bis (B,y-dihydroxy propyl) disulfide, dithiodipropanol, 6, 6-dithiodinicotinic acid, tetramethylthiuram disulfide, cystine, thiamine disulfide, garlic juice disulfide, oxidized glutathione, and mixtures thereof In addition to these disulfide compounds, certain sulfhydryl compounds, which are normally considered enzyme activators, may be used to reversibly inactivate sulfhydryl proteases. These sulfhydryl compounds include mercaptoethanol, cysteine, thioglycerol, dimethyldithio carbamic acid, mercaptopropanol, thionicotinic acid, reduced glutathione and mixture thereof. When these latter compounds are added to an enzyme solution, they undergo air oxidation over a period of several hours to form their disulfide dimer. The level of activity is selected so that the oxidation will be com pleted in a reasonable period of time. Accordingly, mercaptoethanol is oxidized to 2-hydroxyethyl disulfide, thioglycerol is oxidized to bis (B,y-dihydroxy propyl) disulfide, cysteine is oxidized to cystine, etc. The disulfide oxidation product of these compounds is the compound which actually reversibly inactivates the proteolytic enzymes. Of course, the sulfhydryl compound could be oxidized to its disulfide form prior to addition to the enzyme solution.

The above-noted compounds will reversibly inactivate sulfhydryl proteases; however, certain of the disulfide compounds may be more effective when used with one enzyme than another. That is, for a given disulfide inactivator, the rate of inactivation and/or reactivation may vary from one proteolytic enzyme to the next. Thus it has been found that the reactivation rate of papain is generally slower than that of bromelain. For example, 2-hydroxyethyl disulfide will inactivate both bromelain and papain; but using 1 mM glutathione as an activator (see Example 4), the 10 minute reactivation rate is 9.2% for bromelain and less than 1% for pure papain.

Contrary to the utility of the above-listed disulfides, most disulfide compounds are not completely suitable for the preparation of reversibly inactive proteolytic enzyme formulations for ante-mortem injection. This is because most disulfide compounds either will not reversibly inactivate the enzymes, or they may be too toxic, or they result in an inactivated enzyme which reactivates too rapidly in the animal bloodstream. Thus disulfide compounds such as cystamine, methyl disulfide, paramino-phenyl disulfide, 4, 4-dithiodipyridine, 5, 5-dithio-bis-(Z-hitrobenzoic acid) disulfide, dithiodiglycolic acid, dithiodipropionic acid, dithiodibutyric acid, di-n-propyl disulfide, benzyl disulfide, 2-benzothioazoyl disulfide and formamidine disulfide fail to meet all of the requirements of the present invention. Generally, it has been determined that the ineffective disulfide compounds have more electronegative substituents attached to the sulfur atoms than do the effective disulfide compounds.

The reaction of sulfhydryl proteases with the symmetrical disulfide compounds of this invention proceeds in accordance with the following equation:

' Inactivation Activation where ESH is the active sulfhydryl enzyme, such as papain, bromelain, or ficin; RSSR is the symmetrical disulfide inactivator; ESSR is the reversibly inactivated enzyme (a mixed disulfide of the sulfhydryl enzyme); and RSH is a low molecular weight thiol compound formed during the reaction.

The reaction proceeds best at neutral or alkaline pH. Apparently, the proteolytic enzyme (ESH) must be ionized (ES-) beforethe reaction proceeds. The reaction can be driven to completion by oxidation of the thiol (RSH) to the disulfide form by air or with other oxidizing agents. This type of reaction generally proceeds at low temperatures (ice bath temperature) and occurs with virtually no side reactions. Thus, the yields of reversibly inactive enzyme (ESSR) approach 100%.

in the literature, this reaction is generally classified as a thiol-disulfide exchange reaction. This reaction has been known to those in the art for many years, and more recently has been applied to inactivate sulfhydryl enzymes. For example, see Studies on the Active SH Group of Papain by Sanner and Pihl, Journal of Biological Chemistry, Vol. 238, January 1963, and The Scission of the Sulfur-Sulfur Bond by Parker and Kharasch, Chem. Review, Vol. 59, Page 583, 1959. While the reaction itself is well-known, its application to the ante-mortem tenderization of meat is new and unexpected in view of the discovery that only a limited group of disulfide compounds will reversibly inactivate proteolytic enzymes to yield modified enzymes characterized by suitable reactivation rates.

All of the effective disulfide compounds previously mentioned are symmetrical disulfides, i.e. the chemical groups R of the RSSR disulfide are the same. However, by utilizing the thiol-disulfide exchange reaction, a symmetrical disulfide can be converted to a mixed or asymmetrical disulfide:

where R, and R are different. Thus for a given list of thiols and symmetrical disulfide compounds, many combinations of mixed disulfides can be prepared. Depending on the chemical grouping represented by R, the asymmetrical disulfide can be as effective as the symmetrical disulfide in reversibly inactivating sulfhydryl proteases.

The equation for reversible inactivation by an asymmetrical disulfide is as follows:

where R and R are different. Theoretically, both R, and R could form a mixed disulfide with the enzyme ESH. However, in actual practice, one form is usually more stable than the other, and thus predominates.

Special mention should be made of thiamine disulfide, which is non-toxic and has nutritional significance as a vitamin. Thiamine disulfide does not react directly with proteolytic enzymes so as to cause a reversible inactivation. Apparently, thiamine disulfide does not form a mixed disulfide with large molecules, such as pa- 'pain enzyme, and is thus unable to inactivate the enzyme molecule. However, thiamine disulfide will react with low molecular weight thiol compounds to form mixed disulfides. For example, thiamine disulfide will react with glutathione, cysteine, and other naturally occurring thiol compounds to form a mixed disulfide which will inactivate the enzyme protease. In this manner thiamine disulfide can be used to reversibly inactivate sulfhydryl proteases.

It should be clear then that the thiamine disulfide could be first reacted with a low molecular weight thiol, and then the resulting mixed disulfide utilized as the inactivating disulfide RSSR in the above formula. On the other hand, thiamine disulfide can be added directly to crude or commercial grade solutions of enzymes containing a high level of naturally occurring thiol inactivators, such as glutathione. For example, aqueous solutions of crude papain contain high levels of glutathione, and air oxidation of theglutathione would require a long period of time. By adding an excess of thiamine disulfide, usually from about 1 to moles per mole of enzyme in solution, a mixed disulfide is formed between glutathione and thiamine disulfide, which in turn reversibly inactivates the enzyme.

When thiamine disulfide is used in the above manner, i.e. addeddirectly to a crude or commercial grade en zyme solution, it is often desirable to use it in conjunction with another disulfide inactivator. In this way, the thiamine disulfide will tie up the naturally occurring reducing compounds eg glutathione, while the second disulfide reversibly inactivates the protease. In addition to thiamine disulfide, garlic juice disulfides also reversibly inactivate proteases in an indirect manner by first reacting with low molecular weight thiols.

The reactions for indirect inactivation, as with thiamine disulfide, proceed as follows:

where R SSR is thiamine disulfide, R SH is a low molecular weight thiol compound, ESH is the sulfhydryl protease, and RSSR- is a mixed disulfide.

The initial step of the present invention involves preparation of a proteolytic enzyme suspension or solution. Crude enzymes, food grade enzymes and/or relatively pure enzymes may be employed. Food grade enzymes are usually used inasmuch as crude enzymes contain high concentrations of undesirable contaminants, and pure enzymes are relatively expensive to prepare. The reaction may be initiated by adding the disulfide inactivator to a paste suspension of the enzyme prepared by mixing about equal parts of glycerine and enzyme, and then forming a solution by adding cold water to the suspension. Alternatively, the reaction may be initiated by adding the disulfide inactivator to a water solution of the enzyme, or by adding a disulfide inactivator/enzyme powder mixture to water to form a solution. The final enzyme solution may have an enzyme concentration of from about 0.25% to 25% by weight, but enzyme solutions containing about 210% of the enzyme by weight are generally preferred.

Because of the presence of heavy metal ions in most commercial enzyme preparations, it is generally desirable to add chelating agents to the enzyme solution in order to tie up the heavy metal ions, thus lowering their effective concentration. This step is particularly important with crude enzyme powders which contain substantial amounts of interfering metal ions. The presence of a suitable chelating agent, such as salts of ethylenediamine tetraacetic acid (EDTA), helps to assure rapid inactivation of the enzyme. In addition to the salts of EDTA, other suitable chelating agents include citrate salts such as sodium citrate, and phosphate salts such as tetrasodium pyrophosphate, sodium tripolyphosphate, and sodium hexametaphosphate. Also, nitrilotriacetic acid, diethylenetriamine pentaacetic acid, andn-hydroxyethyl amino diacetic acid may be employed. The amount of chelating agent required will of course depend upon the concentration of heavy metal ions present in the enzyme formulation. Generaliy, a chelating agent molarity of from about 0.025 to about 0.3 is suitable.

The enzyme suspension or solution should have a proper temperature and pH in order to obtain an efficient inactivation. The reaction could be carried out at temperatures of from C. to 60C. However, the use of temperatures in the range of 60 may result in irreversible inactivation of the active enzyme fraction. Therefore, it is generally preferred to carry out the reaction within a temperature range of from about 0C. to about 25C., with the most preferred temperature range being from 0C. to 5C. Use of low temperatures inhibits bacterial growth in the enzyme solution, thereby preserving enzyme activity. The pH of the enzyme suspension or solution should be adjusted to from about 6 to 12, and preferably from about 7 to 9, in order that the sulfhydryl enzyme will be ionized to the ES- form. 1n this ionic state, the enzyme will readily react with the disulfide reagent to form a reversibly inactivated mixed disulfide of the sulfhydryl enzyme. It is important that pH of the enzyme solution be adjusted into the desired range soon after preparation of the solution in order that undesirable side reactions are avoided. A pH in the range of 10-12 is generally only used with solutions of papain.

The concentration of inactivating disulfide compounds to be added depends upon several factors, in-

. cluding the concentration of the enzyme in solution,

the inactivator to be used, and the concentration of non-enzyme activator compounds that are present in the enzyme preparation. With so many variables, it is generally necessary to determine experimentally the ratios of inactivator to enzyme required to effect complete reversible inactivation of the active enzyme fraction. Theoretically, one mole of inactivator will react with one mole of enzyme to form one mole of mixed disulfide. However, commercial food grade powders contain non-enzyme sulfhydryl components and metal ions that use up part of the inactivator or interfere with its effectiveness. Hence, molar ratios of inactivator to enzyme of from about I to about 100, and preferably from about 1 to about 10, have been found more adequate for rapid and essentially complete inactivation of commercial enzyme powders. Moreover, an excess of the disulfide inactivator present in the proteolytic enzyme solution will act to tie up glutathione present in the animals blood stream, thus retarding rapid reactivation of the mixed disulfide enzyme.

Once the active enzyme fraction has been reversibly inactivated, the enzyme solution can be injected antemortem into an animal s vascular system. It should be understood that it is the amount of potential enzyme activity injected that is important to controlled tenderization. Generally, it has been found that, depending on the type of animal and the potency of the enzyme preparation, it is necessary to inject from 0.1 to 150 mg. of the enzyme per lb. oflive weight of the animal, with 0.5 to 60 mg. per lb. oflive weight being a preferred range. The animal should be slaughtered within about 1 hour from injection. If the animal is slaughtered too quickly after injection, the enzyme will not have had a chance to be uniformly circulated throughout the animal s vascular system. On the other hand, if the animal is not slaughtered fairly soon after injection, the tenderization effect of the enzyme will not be achieved. Usually it is preferred to dispatch the animal about 10-30 minutes after injection.

The present invention depends on two primary fac tors for success. First, the enzyme must be in a reversibly inactive state at the time of injection. Secondly, the rate of reactivation by reducing agents (mainly glutathione) in the blood must be suitably slow. Of course, the reactivation rate is in part dependent on the level of glutathione in the blood; the higher the level, the faster the reactivation. Glutathione level is known to vary from one animal to another, and from one species to the next. Thus, the rate of reactivation would vary accordingly. We have discovered several techniques for nullifying this blood glutathione factor. As previously mentioned, adding an excess of disulfide inactivator to the enzyme will act to tie up blood gluathione. In addition, we have discovered that by injecting oxidizing or disulfide reagents prior to injection of the enzyme, we could lower the activating capacity of the blood, and thus lower animal reaction. Potassium iodate or tetrathionate function are satisfactory reagents for low ering the activating capacity of the blood, but other reagents work equally well.

The uniqueness and many advantages of the present invention are readily apparent from the following considerations. Firstly, the reaction time required to reversibly inactivate the proteolytic enzyme is greatly reduced. Prior art methods which reversibly inactivate the enzyme by holding the solution at an alkaline pH require from about 12 to 24 hours to achieve inactivation of the enzyme; and many preparations can never be fully inactivated by such treatment. Also, prior art inactivation techniques employing hydrogen peroxide, while relatively fast, result in irreversible inactivation of the originally active enzyme fraction.

Secondly, the present invention results in yields of reversibly inactive enzymes approaching It is practically impossible to obtain these high yields of reversibly inactivate enzyme when the prior art hydrogen peroxide technique is employed, asaddition of hydro gen peroxide causes an irreversible loss of activity.

Thirdly, the present method results in a more stable enzyme solution. The mixed disulfides (ESSR) produced by the present method are resistant to further oxidation and self-digestion (autolysis). Thus, normal enzyme activity losses during enzyme processing (filtering, freezing and storage) are minimized. Storage tests indicate that the enzyme activity of the present disulfide inactivated enzymes is higher than that of companion solutions identical except for absence of disulfide inactivator.

Fourth, and probably most important, the modified enzymes obtained by the present invention are slowly reactivated in the live animal. Animals will tolerate a certain level of active enzyme. If the reactivation rate of the enzyme in the blood is such that this level of active enzyme is not exceeded, an adverse physiological reaction will not materialize. By testing various disulfide inactivated enzymes with various levels of blood glutathione, one practicing the present invention can accurately predict which disulfide inactivator will result in the proper reactivation rate for his particular needs. This control of reactivation rate of the reversibly inactivated enzyme is unique to the present invention. Reversible inactivation by prior art techniques, such as holding at high pH and/or hydrogen peroxide treatment, do not afford any control over the reactivation rate of the enzyme once injected into the animal bloodstream.

EXAMPLE I By definition, a milk clot unit (MCU) is that amount of activity in 1 ml that will cause 5 ml of milk substrate to clot in 60 seconds at C. Thus the formula for calculating the milk clot unit ofa properly diluted enzyme solution is as follows where t=clotting time in seconds: MC U/ml=60/t Separate assay procedures have been used for the determination of reduced (RMCU) and non-reduced (NRMCU) milk clot units. RMCU values are determined using enzyme and milk substrates, both of which are treated with cysteine-versene solution prior to clotting; NRMCU values are determined in the same manner except that the cysteine-versene treatment is omitted. NRMCU values are a measure of active enzyme only, whereas RMCU values measure both active and reversibly inactive enzyme activity. Examples of three typical assays are given in the following Table:

pH RMCU NRMCU Freshly prepared bromelain 4.2 220 125 Aged bromelain" 9.0 186 I7 Tctrathionatc inactivated 9.0 220 0.5

Only water added and analyses made ten minutes later. "pH adjusted to 9.0 and solution held at 15C. for 24 hours.

EXAMPLE 11 In a typicalinactivation procedure, 2.5 millimols of inactivator, 8 grams of enzyme powder, and 8 grams of 5 glycerine were mixed to the consistency of a smooth paste. Separately, a 4 gram quantity of tetrasodium ethylenediaminetetraacetic acid (EDTA) was dissolved in approximately 175 ml of ice cold distilled water containing enough 5N NaOH (predetermined) so that after mixing with the enzyme paste the solution will equilibrate to the desired pH. At time zero, the EDTA-NaOH solution was poured into the ice cold enzyme slurry, and stirred. The resulting mixture was diluted to 200 ml, bringing the enzyme powder concentration to 4%. When assayed at intervals, along with solutions of the same enzyme concentration without EDTA, values were obtained as shown in the following Table:

2 NRMCU VALUES OF 4% BROMELAIN SOLUTIONS AFTER TREATMENT lN 2.5 mM Z-HYDROXYETHYL DlSULFlDE SOLUTION FOR VARlOUS TIME by the disulfide reagent takes place quite rapidly.

EXAMPLE Ill follows:

Rating No physiological reaction detected Increasing degrees of reaction including reddening of comb, lachyrimation, gasping Increasing degrees of severe reaction including gasping, lachyrimation, cyanosis and beginning of a comatose condition The data illustrates the importance of proper pH and the presence of chelating agents in the efficient inactivation of the active enzyme fraction. Disulfide treatment at an acidic pH or without the presence of a chelating agent results in either no inactivation or very slow inactivation. On the other hand, when the enzyme solution contains a chelating agent, and the pH has been adjusted to from 7 to 10, reversible inactivation RMCU/lb Rating (Individual Birds) 0, 0, 0, 0 O, 0 l0 1. l 3.3,4,5.5. 7 9 7 9 3O 9, 9, 9, 10, ii 40 l0 l0, 9

Aging enzyme solutions at alkaline pH generally lowers animal reaction, as indicated in US. Pat. No. 3,446,626 to McAnally. However, addition of cysteine to such solutions prior to injection causes reactivation and a fatal physiological response as indicated in the following experiment where chickens were injected with high levels of enzyme; 40 RMCU/lb for bromelain and 13 RMCU/lb for papain.

Animal Reaction Fresh bromelain 8 Aged bromelain 6 Fresh bromcluin & cysteine 10 Aged bromelain & cysteine Low NRMCU pupain Aged Low NRMCU papuin Low NRMCU papuin& cysteine Aged Low NRMCU pupain &

cysteine Cysteine only 0 This experiment shows that it is the active enzyme fraction which is primarily responsible for adverse animal reactions following ante-mortem injection.

EXAMPLE lV Enzyme solutions were inactivated as outlined in Example II. The excess inactivator was removed by repeated precipitation of the enzyme with ammonium sulfate (40g/ 100 ml) or by passing the solution through Sephadex G25. Then the inactive enzyme was dissolved in pH 7 buffer to give a concentration of 0.3 mM, based on an estimated molecular weight of 18,000.

The reactivation rate was determined by measuring amidase activity using benzoylarginine naphthyl amide (BANA) as a substrate and glutathione (GSH) as activator. A 9.9 ml quantity of 2 millimolar BANA solution in .05 molar pH 7 phosphate buffer was placed in test tubes and equilibrated to 40C. in a water bath. At zero time, 1 ml of 0.3 millimolar enzyme solution was mixed with 1 ml of GSH of the desired concentration, ranging from 1-10 millimolar. Activation of enzyme was carried out at 25C. and terminated by hundred-fold dilution of enzyme-activator mixture at time intervals of 0, l, 2, 4, 6, 8, and 10 minutes. An aliquot of 0.1 ml ofenzyme-activator mixture was transferred into 9.9 ml of BANA solution, and reacted for 15 minutes at 40C. The activity of activated enzyme was measured as described by Blackwood & Mandle (BANA Method), Analytical Biochemistry 2, p. 370-379 (1961)v The enzyme activity was calculated as BANA units per mole of enzyme. As mentioned earlier, the important consideration here is how much active enzyme is released between injection and slaughter. Therefore, the percents of enzymes that have become active in 10 minute periods at 25C. when treated with 1 mM glutathione have been tabulated along with the animal reactivity of the samples.

Percent of Animal inactivator Activated Bromelain" Reaction Control Aged (US. No. 3,446,626) 27.6 7 (l (9) 2-Hydr0xycthyl disulfide 9.2 2 5 (4) Bis (tly-dihydroxy propyl) disulfide 11.2 3 7 (8) Sodium tetrathionatc 12.1 1 8 l2) Tetramethyl thiuram disulfide 17.7 7.4 (5) Cysteine hydrochloride 26.7 62 (4) Cystamine 58.5 8.8 (6) Percent of bromelain that has been reactivated in 10 minutes at 25C. with 1 mM glututhione Injected into chickens at dosage level of 40 RMCUIlb and rated as to degree of animal reaction as described in Example 111. The number of chickens per test is shown in parenthesis.

EXAMPLE V Bromelain solutions inactivated with several different inactivators were injected into chickens at a level of 40 RMCU/lb. At the end of 15 minutes the symptoms were rated, and the time between injection and death was determined for two chickens per sample.

Rating of Animal Survival Disulfidc Reaction Severity Time lnactivator at 15 Minutes Minutes Sodium Tetrathionate 2, 2 28,43

Mercaptoethanol (oxidized to Z-hydroxyethyl disulfidel 2. 3 31,60

Thioglycerol (oxidized to Bis (B,7-dihydroxy propyl) disulfidc 2. 2 35,63

Reuctivated bromelain l0, 10 7,5

Survival time varies from animal to animal. However, even at these high dosages, the above inactivators provided protection for at least 15 minutes.

EXAMPLE VI Groups of six sheep per sample were injected with various enzyme preparations at 40 RMCU/lb. They were dispatched by bleeding twenty minutes after injection. Five organs, namely, heart, gall bladder, mediastinal lymph nodes, kidneys and larnyx were rated on a -10 scale as to severity of reaction. Then the organ rating scores for each aminal were totaled and divided by the number of organs to get an average organ score. Another method of evaluating reactions is to calculate the percent of organs in a given group which were rated or above. The average results were as follows:

In all cases the severity of the animal reaction was reduced by prior treatment of the enzyme with the noted disulfide inactivators. It should be remembered that the level used was substantially higher than required for tenderization.

EXAMPLE Vll To demonstrate the tenderizing effect of properly inactivated enzymes, sheep were injected at a tenderizing dosage. The mutton roasts were cooked by conventional methods and the meat was evaluated for tenderness by an expert panel using a 0-10 scale (l0=excellent tenderness). The average scores for three sheep per bromelain sample are listed in the following table:

Avg. Tenderness Score Non-injected (no enzyme) lnactivatcd by aging at pH 9.0 9.03 lnactivatcd with oxidized mercaptoethanol 9.47

lnactivatcd with Tetrathionate 9.67

The data show that full tenderizing activation was obtained from the disulfide inactivated bromelain samples.

EXAMPLE VllI Separate aliquots of thoroughly mixed ground, lean beef were emulsified in a meat chopper with varying amounts of bromelain which had been inactivated by 60C. for 60 minutes. Cooked sausages were cut into ness with a consistometer. This apparatus consisted of a vertically mounted penetrometer shaft connected to a mechanical pressure gauge and driven vertically at a constant speed by a motor and gear assembly. The shaft terminates in a 7.5 mm flat surface which is forced into the test substance at a constant speed. Resistance to penetration is indicated in arbitrary units on the dial of the gauge.

The resistance of sections of sausages made with varying amounts of enzyme is inversely proportional to the enzyme content, as indicated in the table below. Sausages containing bromelain inactivated with organic disulfides showed even less resistance than anticipated on the basis of the RMCU values, demonstrating their reactivation during the mixing, storage, or cooking operations. lmproved stability of the enzyme which had been inactivated prior to use is indicated by the treatment with various disulfide inactivating agents by lower resistance values of the samples treated with disulfide as compared to enzyme that had not been inactivated, or which had been inactivated with NaOH according to the method of US. Pat. No. 3,052,551. Bromelain inactivated with a non-disulfide agent, e.g. iodo-acetate, is non-reversible, as shown below.

RESlSTANCE OF ENZYME-TREATED & NON-ENZYME-TREATED COOKED BEEF SAUSAGES TO PENETRATION This example shows the effective reactivation, and therefore tenderization, of enzymes reversibly inactivated by treatment with disulfide reagents of the present invention. Conversely, the above data illustrates the treatment of enzymes with other disulfide reagents results in a final product that has not been adequately tenderized, presumably because the enzyme was not sufficiently reactivated in situ.

EXAMPLE IX A. Short Term Stability of Bromelain To determine the maximum amount of activity that can be derived from a given sample of crude enzyme powder, 20 grams of crude bromelain was mixed with 20 grams glycerine in a 250 ml beaker. At time zero,

Treatment pH 71 RMCU NRMCU Water only 4.6 100 100 SN NaOH 5.4 100 94 do. 9.4 83 66 NaOH & EDTA R, Tctrathionate 9.0 100 l The RMCU data show that alkali causes an immediate loss of activity, but that tetrathionate protects against the loss. The NRMCU data show that the bromelain was inactivated in 3 minutes by tetrathionate. Y B. Long Term Stability in addition to short term loss, bromelain solutions are relatively unstable, even at cooler temperature. This is particularly true at pHs of 6-7. To demonstrate this point, bromelain solutions were prepared as outlined in Section A using pH 7 and 9 with and without tetrathionate (all in presence of 5% EDTA). The solutions were stored at 5C. and analyzed at 0, l, 4, 7, l4 and 28 days with the following results.

The data show that the three disulfide inactivators stabilized the bromelain during extended storage. C. Stability Against Peroxide Oxidation Another way to demonstrate protection against oxidation is to add varying amounts of hydrogen peroxide to enzyme solutions that have had various inactivating treatments. Fresh bromelain samples were prepared as in Section A, and mixed for ten minutes before adding peroxide. The peroxide treated samples were stored for one hour at refrigerator temperatures, then analyzed. The results are as follows:

RMCU VALUES Amount Peroxide Fresh Tctrathionatc Treated The results show that fresh bromelain is almost totally destroyed by peroxide, whereas the tetrathionate inactivated sample is virtually unharmed.

EXAMPLE X1 A series of 4% crude ficin solution were prepared with 2% EDTA at pH 9.0. One portion was aged overnite in cooler and the other was inactivated with 10m of either sodium tetrathionate, 2-hydroxyethyl disulfide, or cystamine. The solutions were diluted to contain RMCUfml, and injected at dosage of [ml/lb. (5

RMCU Days Storage at 5C.

pH 7 207 172 133 122 109 104 pH 7 Tetrathionutc 222 214 207 208 214 220 pH) 206 196 176 169 161 161 pH 9 Tetrathionate 207 207 207 200 198 200 RMCU values Bromelain Days lnactivator 0 1 4 7 1 5 Control 208 189 172 165 147 Z-Hydroxyethyl 206 204 200 206 198 disulfide Tetrathionate 206 204 204 203 197 Tetramethylthiuram 207 200 202 207 197 disulfide chickens/group) with the following results:

Average Survival lnactivator Time (Min) Aged (natural inactivation) 13 Sodium tetrathionate Z-hydroxyethyl disulfide 73 Cystamine 9 Both tetrathionate and 2-hydroxyethyl disulfide lowered the animal reaction of iicin, whereas cystamine did not.

EXAMPLE X11 A 1.25% crude papain solution was prepared and divided into portions, and inactivated with several disulfides. The results of injecting lml/lb. in live chickens appear as follows:

Survival lnactivator (lOmM) Time (Min.)

None 34 Tetrathionate l 20* 6,6-dithiodinicotinic acid l20* Thiamine disulfide 77 birds would probably survive indefinitely, but were routinely discarded after two hours.

The results show that all three disulfides lowered the animal reaction of crude papain.

EXAMPLE Xlll zyme injection with the following results:

Avg. Survival Time (Min.)

Control (enzyme only) Potassium ludate & Enzyme Tetrathionatc & Enzyme In another experiment bromelain was inactivated by either 2mM or lmM tetrathionate and injected into five chickens per group with following results:

Avg. Survival Timc (Min.)

2mM tetruthionatc l00mM tctrathionate These experiments indicate that by lowering the activating capacity of the blood by either using an excess of disulfide, or by prior injection of certain chemical oxidizing agents, the animal reaction may be substantially lowered.

EXAMPLE XIV A solution of crude papain was prepared at pH 7.5, divided into three portions and inactivated with 0, 10, or mM thiamine disulfide. NRMCU milk clot values were determined periodically (the solution held in cooler). Following gives rate of drop of NRMCU for the three solutions:

Time Level of Thiamine Disulfidc (Min.) 0 l0mM 20mM 0 36 32 22 l5 33 l 1 I0 32 5 6 45 2 3 6O L5 2 While thiamine disulfide causes the inactivation of crude papain, the fact that the rate of inactivation isn't effected by doubling the level indicates that thiamine disulfide isn't directly inactivating the papain.

EXAMPLE XV Prepared four 5% takamine ficin solutions at pH 7.5 containing 0, 2.5, 5.0 and 10.0% garlic juice. After two 5 hours milk clot values were obtained as follows:

The results indicate that thedisulfide of garlic juice l5 reversibly inactivates crude ficin while protecting the total activity.

Obviously, many modifications and variations of the invention ashereinbefore set forth may be made without departing from the spirit and scope thereof, and thereforeonly such limitations should be imposed as are indicated in the appended claims.

We claim:

l. A method to improve tenderness of meat through proteolytic action, the steps comprising: forming an aqueous solution of a sulfhydryl protease said solution containing from about 0.25 to 25% sulfhydryl protease by weight of the solution; reversibly inactivating the protease by reacting same in the aqueous medium at a pH of from about 6 to 12 and at a temperature of from about 0C. to 25C. with from about 1 to 100 moles of inactivator per mole of sulfhydryl protease, said inactivator selected from the group consisting of 2'- hydroxyethyl disulfide, cystine, oxidized glutathione, bis (By dihydroxy propyl) disulfide, garlic juice disulfides, dithiodipropanol, 6,6-dithiodinicotinic acid, thiamin disulfide, tetramethyl thiuram disulfide, and mixtures thereof; introducing the protease solution into the vascular system of a living animal at a dosage of from aboutO.l to 150 mg. of enzyme per pound of live weight of the animal whereby said protease is slowly reactivated; and thereafter slaughtering said animal.

2. The method of claim I wherein the sulfhydryl protease is selected from the group consisting of plant sulfhydryl proteases, animal sulfliydryl proteases, and microbial sulfhydryl proteases.

3. The method of claim 1 wherein an oxidizing reagent or disulfide reagent is injected into the animals blood stream prior to injection of the sulfliydryl protease solution.

tease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof.

5. The method of claim 4 wherein a chelating agent is added to the reaction medium to tie up heavy metal ions.

6. The method of claim 5 wherein the pH of the enzyme solution has beenadjusted to from about 7 to 9 prior to inactivation.

6 7. The method of claim 6 wherein the disulfide inactivator is thiamin disulfide.

8. The method of claim 6 wherein the animal is subsequently slaughtered within about 1 hour.

9. The method of claim 8 wherein the enzyme solution contains from about 2l0% enzyme by weight of the solution.

10. The method of claim 9 wherein the reaction is carried out at a temperature of from about 0C. to 5C.,

4. The method of claim 1 wherein the sulfliydryl 'proand wherein from about 1 to moles of inactivator are used for every mole of enzyme.

11. The method of claim 10 wherein the enzyme is injected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per lb. of live weight of the animal, and wherein the animal is slaughtered within from about 10 to 30 minutes.

12. A method to improve tenderness of meat through proteolytic action, the steps comprising: forming an aqueous solution of a sulfhydryl protease said solution containing from about 0.25% sulfhydryl protease by weight of the solution; reversibly inactivating the protease by reacting same in the aqueous medium at a pH of from about 6 to 12 and at a temperature of from about 0C. to 25C. with from about 1 to 100 moles of a mixed disulfide inactivator prepared by reacting a thiol compound with a symmetrical disulflde selected from the group consisting of Z-hydroxyethyl disulfide, cystine, oxidized glutathione, bis (13,1 dihydroxy propyl) disulfide, garlic juice disulfides, dithiodipropanol, 6,6-dithiodinicotinic acid, thiamin disulfide, tetramethyl thiuram disulfide, and mixtures thereof; introducing the inactivated protease solution into the vascular system of a living animal at a dosage of from about 0.1 to 150 mg. of enzyme per pound of live weight of the animal whereby said protease is slowly reactivated; and thereafter slaughtering said animal.

13. The method of claim 12 wherein the sulthydryl protease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof. '14. The method of claim 13 wherein a chelating agent is added to the protease solution prior to reaction, and wherein the reaction between the protease and mixed disulfide is carried out at a pH of from about 7 to 9.

15. The method of claim 14 wherein the enzyme solution isinjected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per pound of live weight of the animal, and wherein the animal is slaughtered within from about 10 to 30 minutes thereafter.

16. A method to improve tenderness of meat through proteolytic action, the steps comprising: preparing an aqueous sulfhydryl protease solution at a pH of from about 6 to 12 and at a temperature of from about 0C. to 25C said solution containing from about O.25-25% protease by weight of the solution; adding thereto from about 1 to moles per mole of protease of a sulfhydryl compound selected from the group consisting of cysteine, mercaptoethanol, thioglycerol, mercaptopropanol, thionicotinic acid, reduced glutathione, dimethyl dithiocarbamic acid, and mixtures thereof; holding the protease solution in the presence of air to allow oxidation of the sulfhydryl compound to its disulfide dimer, which in turn reversibly inactivates the sulfhydryl protease; introducing the protease solution into the vascular system of a living animal at a dosage of from about 0.1 to mg. of enzyme per pound of live weight of the animal whereby said protease is slowly reactivated; and thereafter slaughtering said animal.

17. The method of claim 16 wherein the sulfhydryl protease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof.

18. The method of claim 17 wherein a chelating agent is added to the protease solution prior to reaction, and wherein the pH of the protease solution is adjusted to from about 7 to 9 prior to addition of the sulfhydryl compounds.

19. The method of claim 18 wherein the protease solution contains from about 2-10% enzyme by weight of the solution.

20. The method of claim 19 wherein the enzyme is injected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per pound oflive weight of animal, and wherein the animal is slaughtered within from about 10 to 30 minutes thereafter. 

2. The method of claim 1 wherein the sulfhydryl protease is selected from the group consisting of plant sulfhydryl proteases, animal sulfhydryl proteases, and microbial sulfhydryl proteases.
 3. The method of claim 1 wherein an oxidizing reagent or disulfide reagent is injected into the animal''s blood stream prior to injection of the sulfhydryl protease solution.
 4. The method of claim 1 wherein the sulfhydryl protease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof.
 5. The method of claim 4 wherein a chelating agent is added to the reaction medium to tie up heavy metal ions.
 6. The method of claim 5 wherein the pH of the enzyme solution has been adjusted to from about 7 to 9 prior to inactivation.
 7. The method of claim 6 wherein the disulfide inactivator is thiamin disulfide.
 8. The method of claim 6 wherein the animal is subsequently slaughtered within about 1 hour.
 9. The method of claim 8 wherein the enzyme solution contains from about 2-10% enzyme by weight of the solution.
 10. The method of claim 9 wherein the reaction is carried out at a temperature of from about 0*C. to 5*C., and wherein from about 1 to 10 moles of inactivator are used for every mole of enzyme.
 11. The method of claim 10 wherein the enzyme is injected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per lb. of live weight of the animal, and wherein the animal is slaughtered within from about 10 to 30 minutes.
 12. A method to improve tenderness of meat through proteolytic action, the steps comprising: forming an aqueous solution of a sulfhydryl protease said solution containing from about 0.25-25% sulfhydryl protease by weight of the solution; reversibly inactivating the protease by reacting same in the aqueous medium at a pH of from about 6 to 12 and at a temperature of from about 0*C. to 25*C. with from about 1 to 100 moles of a mixed disulfide inactivator prepared by reacting a thiol compound with a symmetrical disulfide selected from the group consisting of 2-hydroxyethyl disulfide, cystine, oxidized glutathione, bis ( Beta , gamma dihydroxy propyl) disulfide, garlic juice disulfides, dithiodipropanol, 6,6-dithiodinicotinic acid, thiamin disulfide, tetramethyl thiuram disulfide, and mixtures thereof; introducing the inactivated protease solution into the vascular system of a living animal at a dosage of from about 0.1 to 150 mg. of enzyme per pound of live weight of the animal whereby said protease is slowly reactivated; and thereafter slaughtering said animal.
 13. The method of claim 12 wherein the sulfhydryl protease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof.
 14. The method of claim 13 wherein a chelating agent is added to the protease solution prior to reaction, and wherein the reaction between the protease and mixed disulfide is carried out at a pH of from about 7 to
 9. 15. The method of claim 14 wherein the enzyme soLution is injected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per pound of live weight of the animal, and wherein the animal is slaughtered within from about 10 to 30 minutes thereafter.
 16. A method to improve tenderness of meat through proteolytic action, the steps comprising: preparing an aqueous sulfhydryl protease solution at a pH of from about 6 to 12 and at a temperature of from about 0*C. to 25*C., said solution containing from about 0.25-25% protease by weight of the solution; adding thereto from about 1 to 100 moles per mole of protease of a sulfhydryl compound selected from the group consisting of cysteine, mercaptoethanol, thioglycerol, mercaptopropanol, thionicotinic acid, reduced glutathione, dimethyl dithiocarbamic acid, and mixtures thereof; holding the protease solution in the presence of air to allow oxidation of the sulfhydryl compound to its disulfide dimer, which in turn reversibly inactivates the sulfhydryl protease; introducing the protease solution into the vascular system of a living animal at a dosage of from about 0.1 to 150 mg. of enzyme per pound of live weight of the animal whereby said protease is slowly reactivated; and thereafter slaughtering said animal.
 17. The method of claim 16 wherein the sulfhydryl protease is selected from the group of enzymes consisting of papain, bromelain, ficin and mixtures thereof.
 18. The method of claim 17 wherein a chelating agent is added to the protease solution prior to reaction, and wherein the pH of the protease solution is adjusted to from about 7 to 9 prior to addition of the sulfhydryl compounds.
 19. The method of claim 18 wherein the protease solution contains from about 2-10% enzyme by weight of the solution.
 20. The method of claim 19 wherein the enzyme is injected into the live animal at a dosage of from about 0.5 to 60 mg. of enzyme per pound of live weight of animal, and wherein the animal is slaughtered within from about 10 to 30 minutes thereafter. 